The adeno-associated virus (AAV) is a non-pathogenic, replication defective virus. Recombinant AAV (rAAV) shows promise as a viral vector for gene therapy. rAAV has a simple structure consisting of 3 capsid proteins and a single-stranded DNA genome. It does not express virally encoded proteins, and is devoid of the integration machinery of wild-type AAV but fortuitously inserts its genetic material into host chromosomal DNA by undefined mechanisms. Despite its non-pathogenic and replication defective nature, there are intracellular virus-host interactions mediated by viral genome DNA (i.e., single-stranded DNA with hairpins). Although there has been substantial progress in the application of this vector system to treat various diseases, the role of intracellular virus-host interactions in rAAV vector biology and host cellular biology in vivo (in animals and humans) remains largely unknown. Our ultimate goal in this proposal is to substantially understand the biological interactions between rAAV vector genomes and host cells in various rAAV-transduced tissues in experimental animals. This is key to selecting appropriate target tissues and understanding the potential risks of rAAV administration as well as designing and developing more efficient and safer gene delivery systems for clinical applications. Based on the observations by us and others, we hypothesize that rAAV genomes activate various DNA repair pathways via virus-host cell interactions, and trigger rAAV-rAAV and/or rAAV-host genome recombinations. We have recently demonstrated that a specific DNA repair endonuclease opens the rAAV vector genome inverted terminal repeat (ITR) hairpin structures and trigger rAAV genome recombinations. In the project, we will first identify cellular factors involved in rAAV genome processing and investigate their course of action (aim 1). Second, we will investigate how rAAV genomes and the cellular factors interact in a network and how rAAV vectors activate this network (aim 2). Finally, we will characterize and quantify rAAV integration in various tissues in mice and will identify cellular factors involved in rAAV integration to understand the mechanisms for rAAV integration in vivo (aim 3). The proposed project should substantially contribute to building an intellectual foundation for successful human gene therapy. Public Relevance Statement: Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector system for human gene therapy. This proposed project aims at elucidating complex interactions between rAAV vector genome, the host cellular genome and cellular proteins in vector-infected cells for a better understanding of this vector system. Results of the study will contribute to designing and developing more efficient and safer rAAV vectors and assessing the safety of the vector. [unreadable] [unreadable] [unreadable]